In vivo imaging of hepatobiliary metabolism, transport, and hepatic morphology. Utilizing endogenous tissue autofluorescence in tandem with exogenous fluorescent probes allowed in vivo elucidation of biological structure and function. Illustrated is the in vivo application of 7-benzyloxyresorufin (7BR) for detection of CYP3A metabolic activity. Dechorionated embryos exposed (aqueous bath) to the CYP3A substrate 7BR exhibited 7-benzyloxyresorufin-O-dealkylation (BROD) activity, which resulted in the generation of the fluorescent metabolite resorufin (red). All in vivo images from an individual STII medaka, 6 dpf. (A) Brightfield microscopy, showing liver (L) and gall bladder (GB). (B1) Same animal as in frame A, imaged with widefield fluorescence microscopy (DAPI/UV excitation) illustrating tissue autofluorescence. (B2) Widefield fluorescence (TRITC) image capture of resorufin (indicative of CYP3A metabolic activity) fluorescence in the intrahepatic biliary passageways of the embryonic liver. Resorufin fluorescence is distinct and limited to the intrahepatic biliary passageways of the liver (L) and gall bladder (GB). (B3) Color composite of B1 and B2, DAPI/TRITC image captures, illustrating resorufin fluorescence (red) in the liver (L) and gall bladder (GB). (C1 and C2) In vivo imaging of region of interest in frame B3 (white square) showing in vivo phenotype of hepatic parenchyma at 6 dpf. Six to 8 hepatocytes were observed (in transverse section) to surround a central bile passageway (IHBP) at their apical membranes. C2 illustrates concentrative transport of resorufin from hepatocellular cytosol to intrahepatic biliary passageways (IHBPs), indicated by increased fluorescence in the tubule lumen, or intrahepatic biliary passageway. Red blood cells were observed actively circulating through hepatic sinusoids (S/r). Hepatocyte nuclei (HN).